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BIO1140 (682)


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University of Ottawa
Kathleen Gilmour

Transcription -DNA is organized into functional DNA and non-functional DNA (genic and non-genic) Functional Unit- describes what is going to be transcribed from DNA to RNA -transcription involves nucleoside triphosphates instead of deoxynucleoside triphosphates (as found in replication) -transcription can start de novo -RNA polymerases do not need a primer as long as there is some regulatory system to tell it where to start -the first nucleotide that is added will have a triphosphate on it -the other nucleotides will have monophosphate Genes -a gene is described as something the codes for proteins that is less than 100 aa long (according to genomics) -originally, its definition was genetic and reflected the lack of understanding of DNA or RNA -e.g.: a gene is a unit associated with an inherited trait -e.g.: genes are the units of heredity in living organisms -e.g.: genes are the biological or basic unit of heredity found in all cells -later, a gene was defined with molecular information -e.g.: a gene is a unit of information on a chromosome -e.g.: a gene is a basic unit of heredity; a section of DNA coding for a particular trait -in order to make a sense strand, you need to synthesize from the anti-sense strand -the genetic code is redundant -mutations can be made and may not affect some things -amino acids are coded for by more than 1 codon -genes have introns; if mutations occur in introns, it is removed during splicing Modern View of a Gene and Chromosome -made up of exon, intron, exon, intron (which is removed), a TATA box (an A-T rich sequence; the point at which RNA polymerase binds to start transcription), promoter (proximal; sequences that regulate and is close and upstream to where RNA polymerase binds), and promoters that are further away known as enhancers (distal promoters) found on either side -sequences that bind proteins needed fro transcriptional control and RNA synthesis are cis-acting -refers to usually a regulatory sequence that usually bind proteins -these proteins are said to be trans-acting -a cis-regulatory element or cis-element is a region of DNA or RNA that regulates the expression of genes located on that same molecule of DNA (often a chromosome) -these cis-regulatory elements are often binding sites for one or more trans-acting factors -a gene can be described as a basic unit of hereditary material -it is an ordered sequence of nucleotide bases that encodes a product -the gene includes regions preceding and following coding regions as well as intervening sequences (i.e.: introns) between individual coding segments (i.e.: exons) -the simplified idea of "one gene - one protein - one structure - one function" cannot take into account proteins that have multiple functions depending on context -most of the genome in higher eukaryotes consists of repeated sequences and mobile elements called transposons or retrotransposons (retroposons) -only a few % code for genes; the protein coding regions are only ~1.5% of the total -transposable elements involve elements that can make a copy of themselves, independent of the genome replication, and then insert somewhere else Genomics -refers to activities at the DNA level (i.e.: sequences) -includes cloning and functional analysis -functional genomics describes what the genes do Epigenomics -looks at DNA modifications that are heritable and can influence the phenotype (i.e.: 5 Methyl-C) Transcriptomics -looks at the transcriptomes via the RNA by converting it to DNA and sequencing -is simple Proteomics -looks at all the proteins -can only look at ~1000 at a time and is very time consuming Transcription -in prokaryotes, there is no compartmentalization for transcription, replication, or translation -in eukaryotes, the message that is made is not the final sequence that is translated -general characteristics of RNA: -usually single-stranded (one polynucleotide) but with double stranded regions -complimentary to part of a DNA polynucleotide -has ribose instead of deoxyribose -has uracil instead of theymine -may contain modified bases -made by RNA polymerases instead of DNA polymerases -1 type of RNA polymerase in prokaryotes -is a multi-enzyme complex -changing sigma-factors allows for recognition of different classes of enzymes -3 or more types of RNA polymerase in eukaryotes (3 in the nucleus and 1 or more in the mitochondria) -allows for specialization of the polymerase -the cis-acting sites that are recognized by the polymerase are different for each type of polymerase -different RNA polymerases transcribe different sets of genes -RNA polymerase must recognize different signals in the DNA to recognize the correct gene to transcribe -RNA polymerase I transcribes rRNA in the nucleolus (part of the nucleus) -RNA polymerase II transcribes mRNA (encodes proteins) and most snRNAs (small nuclear RNA) -RNA polymerase III transcribes tRNA, 5S rRNA, some snRNAs amd scRNAs (small cytoplasmic RNA) -RNA polymerase starts at the 5' end and the regulatory sequences are adjacent; the promoter is at the 5' end -promoters are in the middle of the gene for some tRNA and 5S rRNA -overview of transcription: 1) starts when RNA polymerase binds to DNA and DNA has to open up (will require helicase; may or may not need topoisomerase, depending on its length) 2) DNA double helix begins to unwind 3) synthesis from 5' to 3'; RNA polymerase adds RNA nucleotides sequentially according to the DNA template -the template is read from 3' to 5' -the point of reference is the strand that is being synthesized -this strand is the coding strand -this is the sense strand - '+' strand for viruses -the template strand is the non-coding strand -anti-sense strand - '-' strand for viruses 4) once the RNA is completed, it can be removed from the template -in eukaryotes, it is processed and then exported to the cytoplasm (unless it is a snRNA, which is processed but remains in the nucleus) -in prokaryotes, the message is released and goes to the ribosomes or translation can be initiated while transcription is occurring -before transcription is complete, you can initiate again using another polymerase -allows for multiple transcripts being read from the same gene at the same time promoter- controls the sequences that initiates transcription transcription unit- the portion of the gene that is copied into RNA -the RNA can be coding or non-coding -if it is coding, only a part of the message has the open reading frame that is going to be translated terminator- signals the end of transcription of a gene -RNA polymerase catalyses the polymerization of ribonucleoside 5'-triphosphates (rNTPs or NTPs) and makes a monophosphate chain (except at the 5' end where the triphosphate remains) -the triphosphate site at the 5' end acts as a specific site for modification -triphosphates are added until a termination signal is received -RNA polymerase does not require a primer; transcription is initiated de novo -RNA polymerase initiates synthesis at promoter sequences on DNA, upstream (5') to the transcription start site -other sequences such as enhancers can affect transcription -RNA polymerase adds (NTPs) at the 3' end of a new polynucleotide until it encounters a termination signal at a defined sequence structure
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